The broad goal of this proposal is to understand regulation and molecular details of proteolysis-independent ubiquitination. Ubiquitination of proteins has been recognized as an important regulatory mechanism. Highly conserved multi-protein complexes called SCF play a central role in many ubiquitination processes. SCF-mediated ubiquitination of target proteins usually initiates their proteolysis by the 26S proteasome. The investigator has recently discovered that SCF-mediated ubiquitination can directly regulate protein function without proteolysis. Specifically, SCFMet3O catalyzes oligoubiquitination of the transcription factor Met4. Ubiquitination of Met4 blocks the formation of protein-protein interactions with other transcription factors and ultimately blocks assembly of a functional transcription complex. Compared to the role of SCF in targeting proteins for proteolysis, little is known about this proteolysis-independent function of SCF. However, regulation of protein function by ubiquitination without proteolysis seems to be an important and among eukaryotes highly conserved process. To learn more about this process the applicant will utilize the well characterized Met4 oligoubiquitination as a model. Similar to Met4, the yeast kinetochore component Ndc10 might also be regulated by proteolysis-independent ubiquitination. Sequence comparison revealed that Met4 and Ndc10 share a highly conserved region. Furthermore, it has been shown that Ndc10 is ubiquitinated but not degraded. Interestingly, Met4 and Ndc10 ubiquitination depend on the same ubiquitin conjugating enzyme, namely Cdc34. Therefore, in addition to the studies on Met4 he will analyze Ndc10 ubiquitination and its implication on kinetochore function. Specifically, the experiments proposed are aimed to (1) map modification sites in Met4; (2) identify components of the enzymatic machinery and the signal transduction pathway that initiate ubiquitination and deubiquitination of Met4: (3) understand how the length of the ubiquitin chain on Met4 is regulated; and (4) analyze the biological relevance and the ubiquitination machinery that oligoubiquitinates Ndc10. In each case the powerful combination of yeast molecular and genetic approaches will be applied. Yeast has proven to be a successful model system to study ubiquitination. It is therefore anticipated that the proposed research together with studies in other experimental systems will contribute to the understanding of regulation of protein function by ubiquitination. This might eventually be important in diagnosis and treatment of human diseases.